European Space Agency - Granada Crystallisation Facility (ESA-GCF) - 11.22.16
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This investigation tested an apparatus for crystallizing a variety of protein solutions in space and on the ground. Most protein molecules crystallized by the apparatus showed quality comparable to other techniques. Analysis revealed differences depending on conditions such as medium, temperature, and ground versus space. Early results showed no difference in free protein solutions in space and gel solutions on the ground, although problems with temperature profiles prevent direct comparison. On a subsequent mission, ground temperature was more stable than on the station, which again precluded comparisons. The apparatus accidentally spent an extra four weeks in space, which also could have affected results. Experiment Details
Juan Manuel Garcia-Ruiz, Ph.D., University of Granada, Granada, Spain
Luigi Carotenuto, MARS Center, Naples, Italy
Dario Castagnolo, MARS Center, Napoli, Italy
E. Manas, University of Granada, Granada, Spain
Triana Science and Technology, Granada, Spain
Sponsoring Space Agency
European Space Agency (ESA)
ISS Expedition Duration
August 2001 - December 2001; June 2002 - December 2002
Counter-diffusion method for protein crystallization works on allowing the protein and the precipitating agent solutions to diffuse one into each other (Figure 1). Since the salt molecules diffuse faster than the biological macromolecules (typically one to two orders of magnitude faster) and due to the coupling between mass transport and precipitation, a wave of supersaturation is triggered which moves across the protein chamber provoking successive precipitation phenomena at different crystallization conditions. Unlike classical techniques, the counter-diffusion technique self searches for the best crystallisation conditions by exploring a large number of crystallization conditions in one single experiment, thus reducing drastically the screening for crystal quality. An additional advantage is that it can be implemented inside X-ray capillaries, avoiding post-crystallisation handling of crystals. It has proven to perform very well on the ground using slightly gelled protein solutions to prevent natural convection. It is obvious that because of the very nature of the technique, a purely diffusive mass transport is a requisite to implement counter-diffusion techniques. On the ground, the experiments must be performed with the help of thin (100 microns in diameter) capillaries or gel solutions, two techniques that present certain drawbacks. The best scenario for this type of study seems to be in weightless conditions. In order to prove this, three experiments were carried out with APCF crystallization boxes during the STS-95 US space shuttle mission, to test reverse diffusion crystallization techniques. The data was measured with a Mach- Zehnder interferometer and was consistent with the behaviour observed in simulations and experiments on the ground. On the basis of these observations, an apparatus -Granada Crystallization Box (GCB)- was designed with the aim of optimising the number of experiments, of reducing the volume of protein solutions, making easier the implementation of the apparatus and reducing the cost of the space crystallisation. The main objective of our experiment was to validate the concept and functioning of this apparatus, specifically designed to be used in space experiments. Each of the GCBs contained a single protein along with its precipitating agent and additives. Different proteins were used, selected by LEC from proposals submitted by European and Russian laboratories. The starting conditions of the experiment were set in previous pre-flight experiments. By its very nature, counter-diffusion techniques work only under diffusion controlled mass transport. Thus, the second objective of the project was to compare identical experiments performed in space with free protein solutions on one hand, and on the ground in gelled protein solutions on the other hand. In order to evaluate the differences between a pure diffusive environment on-earth (achieved in highly viscous protein solution made with agarose at 0.1% w/v) and an agarose-free scenario provided by ISS, where sedimentation and convection are not completely removed due to residual accelerations. This objective was addressed with a fluid dynamics study of crystallisation in capillaries of different diameter and a 2D simulation of the counter-diffusion technique.
Operational Requirements and Protocols
Crystallisation experiments were performed in Granada Crystallisation Box (GCB). GCB is a device to perform counter-diffusion experiments designed and developed by LEC. The concept stems on research in process modelling and optimisation sponsored by ESA. GCB consists of 3 elements made of polystyrene: A reservoir to introduce the gel, A guide to hold capillaries, A cover. The buffer layer is located at the bottom of the reservoir after inserting the guide. Once the gel is set, the capillaries are filled with the protein and inserted trough the hole of the guide and punctuated to a given depth into the gelled buffer layer. Finally the salt solution is set on top of the buffer gel. Each of the boxes accommodates six capillaries with a maximum inner diameter of 1.5 mm. The external dimensions of the GCB are 33 x 100 x 7 mm. The weight of one GCB filled with six capillaries and the chemical solutions is 22 grams. Obviously, Granada Crystallison Boxes cannot be used directly in space, because of safety reasons. It was necessary to enclose each GCB in plastic bags and then into a container designed ad-hoc. This external container is the Granada Crystallisation Facility. The Granada Crystallisation Facility (GCF) consists of an aluminum container coated with an electroless nickel plate-PTFE finishing. The inner walls of the GCF base are covered by a foam and lined with an absorbent polypropylene-cellulose tissue . The container has external dimensions of (134 x 134 x 84) mm3. The external dimensions of the GCF were constrained by the weight allocated in the Andromede mission to this experiment (1.4 kg) and by the dimensions of the location that RSC-Energia assigned to the GCF in the ISS and in the return Soyuz vehicle. The GCF can host up to twenty-three GCBs (that is, a maximum of 138 capillaries) plus a temperature recorder. Each GCB was enclosed inside a thermo-sealed polyethylene-polyamide bag and placed in the container along with the corresponding separation foam pads. In addition, a temperature miniature data logger [(31x31x31) mm3] (from Meilhaus Electronic) can be loaded in the container. This data logger, called Sugar Cube, is an autonomous working device containing a specific sensor for measurements of temperatures. The measuring period is programmable between 1 second and 24 hours with a precision of one second, and the acquired data are stored internally. Each data logger contains an infrared interface (IrDA) for the wireless communication and data exchange with PC or laptop. Each GCB was used for a single protein with identical starting conditions but different inner diameter of the capillaries and/or different protein concentration. The total weight of the GCF ready for the flight experiment is 1.2 kg.
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Once a first inspection study performed at LEC laboratories was finished, the samples were sent to their corresponding investigators, along with a first analysis report and a document giving them some suggestions for storage and X-ray data collection, since the crystals were agreed to be property of the laboratories supplying the proteins and these laboratories were also responsible of the evaluation of the crystal quality.
During the first inspection study above mentioned, the location of the crystals inside the capillaries was studied, noting whether these were influenced by capillary diameter, and comparing them with the experiments performed on ground.
Most of the molecules did crystallize, both in space and on ground, and correspond to those molecules which crystallization conditions were adequately optimised. Owing to the very short time available to prepare this project, the optimal crystallization conditions could not be found for some molecules, and they did not crystallize, either in space or on ground.
X-ray diffraction analysis was the task of the laboratory proposing and supplying each particular protein. Crystals from equivalent positions and grown in capillaries with the same diameter were selected and aligned in a similar orientation. Data collection was carried out at beam lines BW7B and X13 (EMBL-Hamburg Outstation). Crystals grown in microgravity and their on-ground counterparts were flash-cooled at 100 degrees K and some images 90 degrees apart were collected and auto-indexed with Mosflm. Large crystals of thaumatin (rod-like) were collected at room temperature without any further manipulation. The collection of the data set was carried out on the basis of the best strategy predicted by Mosflm. Special attention was paid to the estimated redundancy and oscillation angle since I/sigma(I) is dependant on both. Images were recorded on a Mar 345 image plate (beam line BW7B) and a Mar CCD (beam line X13) detectors. Data reduction was carried out with XDS (Kabsch, 1993).
Both on ground and microgravity grown crystals show very good I/sigma(I) values. For the model proteins the values are comparable to the best obtained by other techniques. In the case of lysozyme, the data set was collected at 0.95 Angstrom but many spots at the border of the detector had I/sigma(I)>2 and the crystal may diffract well beyond 0.95 Angstrom. The resolution limit has been improved for dehydroquinase and catalase from 3.50 and 3.40 Angstrom to 1.71 and 1.60 Angstrom respectively.
An analysis of the slight differences in crystal quality in terms of I/sigma(I) as a function of the resolution, reveals the inexistence of a clear and consistent pattern that leads to conclude which environment yields the best crystals. A deeper analysis of the data shows that the best crystals from dehydroquinase and HEW lysozyme are those that have larger multiplicity regardless their precedence. For catalase, similar redundancies yield similar crystal quality. The effect of redundancy on I/sigma(I) is clearly observed in the thaumatin: data sets collected at room temperature from crystals grown in space with gel, without gel and on ground share similar multiplicity and yield similar crystal quality in terms of I/sigma(I). Interestingly, the differences in crystal quality that appear between the data sets collected at 100 Kelvin and their counterpart collected at room temperature can be correlated with differences in redundancy.
It is important to emphasize the kind of comparison between crystal grown on ground and crystal ground in space in our experiment. In most previous space crystallisation studies, the comparison is made between crystals grown under convective conditions on ground and putative convection-less conditions in space (references). Unlike this, we are here comparing a) crystals grown under perfect diffusive (convection-less) conditions on ground due to the use of agarose, and b) crystals grown in space under convective-diffusive conditions in space. Therefore, it is not surprising the similarity in crystal quality between these two types of scenarios.
By its very nature, counter-diffusion techniques work only under diffusion controlled mass transport. Thus, the second objective of the project was to compare identical experiments performed in space with free protein solutions on one hand, and on the ground in gelled protein solutions on the other hand. In order to evaluate the differences between a pure diffusive environment on-earth (achieved in highly viscous protein solution made with agarose at 0.1percent w/v) and an agarose-free scenario provided by ISS, where sedimentation and convection are not completely removed due to residual accelerations. This objective was addressed with a fluid dynamics study of crystallisation in capillaries of different diameter and a 2D simulation of the counter-diffusion technique.
In some cases, crystal quality was evaluated by X-ray diffraction. Regarding the crystal quality, the preliminary analysis of the data sets collected with synchrotron radiation shows that the crystals grown with the counter-diffusion technique share excellent global indicators of x-ray data quality with no obvious difference between crystals grown under reduced convection conditions in space and crystals grown under convection free conditions on ground.
It is noticeable that due to unexpected logistic and safety problems we were unable to use the data recorder with the space GCF. Therefore it is impossible to know if the temperature profile during the mission and the one on the GCF on ground were similar enough to permit a reasonable crystal quality comparison.
During the Odissea mission fifteen different protein solutions supplied by European and Japanese laboratories were used to prepare twenty three GCB boxes that were accommodated in the GCF and flew to the ISS on September 25, 2002.
For comparative studies, one additional GCF was prepared in parallel on-ground. The unique difference between the space and on-ground experiments were that for on-ground experiment the viscosity of the protein solution was increased by adding agarose at concentration of 0.1 percent. These on-ground experiments were prepared in Baikonur at the same time that the space experiment and kept at LEC laboratories in Granada, Spain. In order to record the temperature profile during the experiment, a small temperature sensor/recorder was located inside the GCF (the so called Sugar Cube from Meilhaus Electronic, GmbH, Germany). During this mission, the facility stayed 74 days in space.
After the flight all the GCB were carefully inspected. It was confirmed that none of the 23 GCB suffered any damage. The crystallisation patterns inside the capillaries were photographed with the help of a binocular microscope.
Once a first inspection study performed at LEC laboratories was finished, the samples were sent to their corresponding owners, along with a document including the final conditions for each particular experiment and a report including the results of the inspection that we performed in our laboratory of the on ground experiment at the experimentation corresponding time equivalent to the time at which the samples were located at the ISS. A CD with a selection of the photos we took of space and on-ground experiments was also sent to all the participant laboratories.
It is clear that the temperature on-ground was much more stable than in the ISS. In fact, the temperature values in the location of the GCF at the ISS had many fluctuations during the mission. Temperatures gap of up to eight degrees Celsius (14 to 22 degrees C) were recorded.
One of the objectives of the experiment was the comparison of the crystallization process on ground and in space in terms of crystal quality and crystal morphology and pattern of precipitation. Because of the very different temperature profile between the space and on-ground variation, it was impossible to perform properly these studies.
Unfortunately, an additional problem occurs during the mission. The GCF located at the ISS returned Earth later than the date on schedule because it was leaved in the ISS when the Soyuz spacecraft returned to Earth. Four weeks later the GCF returned to earth on board an American space shuttle. This extra time and the handling of the GCF inside the ISS from Russian to American modules and accommodation in the Shuttle could have affected the experimental results.
Evrard C, Maes D, Zegers I, Declercq J, Vanhee C, Martial J, Wyns L, Van de Weerdt C. TIM Crystals Grown by Capillary Counterdiffusion: Statistical Evidence of Quality Improvement in Microgravity. Crystal Growth and Design. 2007 November; 7(11): 2161-2166. DOI: 10.1021/cg700687t.
Maes D, Gonzalez Ramirez LA, Lopez-Jaramillo J, Yu B, De Bondt H, Zegers I, Afonina E, Garcia-Ruiz JM, Gulnik S. Structural study of the type II 3-dehydroquinate dehydratase from Actinobacillus pleuropneumoniae. Acta Crystallographica Section D: Biological Crystallography. 2004; Section D60: 463-471. DOI: 10.1107/S090744490302969X.
Maes D, Decanniere K, Zegers I, Vanhee C, Sleutel M, Willaert R, Van de Weerdt C, Martial J, Declercq J, Evrard C, Otalora Munoz F, Garcia-Ruiz JM. Protein crystallisation under microgravity conditions: What did we learn on TIM crystallisation from the Soyuz missions?. Microgravity Science and Technology. 2007; XIX-5/6: 90-94.
Ground Based Results Publications
ESA Erasmus Experiment Archive
Example of proteins crystallized in the Andromede mission a) Lysozyme; b) Dehydroquinase; c,e) Thaumatin; d) Concanavalin A; f,k,n) Anti lysozyme camel antibody; g) Insulin; h) Lumazine synthase; i) Catalase; j,o) Factor XII; l) Saicar synthase; m) Ferritin. Images are courtesy of LEC, Granada, Spain.
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Example of proteins crystallized in the GCF in the ISS. Images are courtesy of LEC, Granada, Spain.
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Example of proteins crystallized in the GCF on- ground. Images are courtesy of LEC, Granada, Spain.
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